Abstract 16598: Carnosine Synthetase Overexpression in Skeletal Muscle Promotes Functional Recovery of Ischemic Limb

Abstract

Carnosine synthetase (ATPGD1) is an ATP grasp enzyme that catalyzes formation of histidyl dipeptides such as carnosine (β-alanine-histidine) and anserine (β-alanine-methyl histidine). These dipeptides are food constituents found in a high concentration in the skeletal muscle (10-20 mM) that have the abilities to chelate metals, scavenge lipid peroxidation products and buffer intracellular pH. These dipeptides are regarded as protective against injury and stress. Despite this knowledge, whether carnosine regulates angiogenesis is unknown. To assess this potential, a model of hindlimb ischemia (HLI) was surgically induced by femoral artery and vein ligation in the C57BL/6J (wild type, WT) mice provided drinking water without or with carnosine (1g/L) for 21 days post-surgery. C57BL/6J WT mice subjected to HLI and supplemented with carnosine had increased skeletal muscle expression of the dipeptide/histidine transporter (PHT1), carnosine transport, improved blood flow recovery, re-vascularization and regeneration of myocytes in the ischemic limb compared with mice without carnosine. To support that elevated skeletal muscle carnosine was beneficial in HLI, transgenic mice overexpressing ATPGD1 in the skeletal muscle were constructed. Transgenic (α-HSA-ATPGD1TG) mice had ~2-2.5-fold increase in carnosine (WT: 2.71±0.54 vs 7.40±0.54 nmoles/mg protein α-HSA-ATPGD1TG; p <0.05) and anserine levels (WT: 3.65±1.12 vs 8.04±0.23 nmoles/mg protein α-HSA-ATPGD1TG p <0.05) in the skeletal muscle than WT mice. At 2 and 3 weeks post-HLI surgery, α-HSA-ATPGD1TG mice had significantly improved blood flow recovery compared with the WT mice (2 weeks: α-HSA-ATPGD1TG: 59±3% vs 45±3% WT; p <0.05; 3 weeks: α-HSA-ATPGD1TG: 65±4% vs 46±2% WT; p <0.05). Because faster recovery of blood flow in the carnosine-supplemented mice was accompanied with increased expression of hypoxia inducible factor (HIF-1α) and vascular endothelial factor (VEGF) in the ischemic limb, we further investigated the mechanistic connection. Skeletal muscle myoblasts C2C12 cells pretreated with octyl-D-carnosine and subjected to 1 h of hypoxia, replacing oxygen with 95% N 2 and 5% CO 2 , had enhanced HIF-1α translocation, VEGF mRNA and secretion compared with untreated hypoxic and normoxic cells. Collectively, our results demonstrate that carnosine improves ischemia-stimulated angiogenesis potentially by stabilizing HIF1-α and promoting VEGF formation suggesting that carnosine may be a useful therapy in critical limb ischemia.